Determination of precise and accurate protein structures by Nuclear Magnetic Resonance (hereinafter “NMR”) generally requires weeks or even months to acquire and interpret all of the necessary NMR data, particularly for completing side chain resonance assignments. However, medium-accuracy fold information can often provide important and helpful clues about protein evolution and biochemical function(s). A largely automatic strategy for rapid determination of medium-accuracy protein backbone structures has been previously described (Zheng, D., Huang, Y. J., Moseley, H. N. B., Xiao, R., Aramini, J., Swapna, G. V. T.; Montelione, G. T. (2003) Automated protein fold determination using a minimal NMR constraint strategy. Protein Science 2003, 12: 1232-1246.). This strategy of rapid fold determination derives from ideas originally introduced for determining medium-accuracy NMR structures of large proteins (Gardner, K. H., Rosen, M. K., and Kay, L. E. (1997). Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR. Biochemistry 36, 1389-1401.), using deuterated, 13C—, 15N-enriched protein samples with selective protonation of sidechain methyl groups (13CH3). Data collection includes acquiring NMR spectra suitable for automatic analysis of assignments for backbone and sidechain 15N, HN resonances, and sidechain 13CH3 methyl resonances. In some cases, assignments are also determined for 1H and/or 13C atoms of labeled Tyr and Phe residues. NMR resonance assignments can be determined by automated NMR assignment programs, such as the program AutoAssign (Moseley, H. N., Monleon, D., and Montelione, G. T. (2001). Automatic determination of protein backbone resonance assignments from triple resonance nuclear magnetic resonance data. Methods Enzymol 339, 91-108; Moseley, H. N., and Montelione, G. T. (1999). Automated analysis of NMR assignments and structures for proteins. Curr Opin Struct Biol 9, 635-642; Zimmerman, D. E., Kulikowski, C. A., Huang, Y.; Feng, W., Tashiro, M., Shimotakahara, S., Chien, C., Powers, R., and Montelione, G. T. (1997). Automated analysis of protein NMR assignments using methods from artificial intelligence. J Mol Biol 269, 592-610; Zimmerman, D. E., and Montelione, G. T. (1995). Automated analysis of nuclear magnetic resonance assignments for proteins. Curr Opin Struct Biol 5, 664-673). Three-dimensional structures can be analyzed automatically with programs like AutoStructure (Huang, Y. J., Moseley, H. N., Baran, M. C., Arrowsmith, C., Powers, R., Tejero, R., Szyperski, T., and Montelione, G. T. (2005). An integrated platform for automated analysis of protein NMR structures. Methods Enzymol 394, 111-141; Huang, Y. J., Tejero, R., Powers, R., and Montelione, G. T. (2006). A topology-constrained distance network algorithm for protein structure determination from NOESY data. Proteins 62, 587-603). The total time required for collecting and processing NMR spectra for the medium-accuracy strategy can be relatively short. For example, using NMR data on 2H, 13C, 15N-enriched proteins with protonated methyl (and/or aromatic) groups, published NMR software packages like AutoAssign and AutoStructure can be used to process NMR spectra, carry out resonance assignments, interpret Nuclear Overhauser Enhancement Spectroscopy (hereinafter “NOESY”) data, and generate medium-accuracy structures within a few days. These structures provide essential three-dimensional information for characterizing biological activities of proteins, and are good starting points for further refinement to high precision and accuracy using additional NMR data. The feasibility of this combined data collection and analysis strategy starting from raw NMR time domain data has already been demonstrated by automatic analysis of a medium accuracy structure of the Z domain of Staphylococcal protein A (Zheng, D., Huang, Y. J., Moseley, H. N. B., Xiao, R., Aramini, J., Swapna, G. V. T.; Montelione, G. T. (2003) Automated protein fold determination using a minimal NMR constraint strategy. Protein Science 2003, 12: 1232-1246).
Perdeuteration (the enrichment of proteins with 2H) is also a prerequisite for using some of the most advanced NMR methods for studying the three-dimensional structures of membrane proteins by NMR (Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001). Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8, 334-338; Fernandez, C., Hilly, C., Wider, G., Guntert, P., and Wuthrich, K. (2004). NMR structure of the integral membrane protein OmpX. J Mol Biol 336, 1211-1221; Fernandez, C., Hilty, C., Wider, G., and Wuthrich, K. (2002). Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy. Proc Natl Acad Sci USA 99, 13533-13537; Fernandez, C., and Wuthrich, K. (2003). NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Lett 555, 144-150; Sorgen, P. L., Cahill, S. M., Krueger-Koplin, R. D., Krueger-Koplin, S. T., Schenck, C. C., and Girvin, M. E. (2002a). Structure of the Rhodobacter sphaeroides light-harvesting 1 beta subunit in detergent micelles. Biochemistry 41, 31-41; Sorgen, P. L., Hu, Y., Guan, L., Kaback, H. R., and Girvin, M. E. (2002b). An approach to membrane protein structure without crystals. Proc Natl Acad Sci USA 99, 14037-14040; Tamm, L. K., Abildgaard, F., Arora, A., Blad, H., and Bushweller, J. H. (2003). Structure, dynamics and function of the outer membrane protein A (OmpA) and influenza hemagglutinin fusion domain in detergent micelles by solution NMR. FEBS Lett 555, 139-143)). These methods require use of 2H, 13C, 15N-enriched membrane protein samples with 13C—1H (or 12C—1H) methyl labels. Production of such samples can be expensive ($1,500-$10,000 per sample), limiting the applicability of this approach. The high cost of sample production greatly limits the applicability of powerful automated structure analysis methods (Zheng, D., Huang, Y. J., Moseley, H. N. B., Xiao, R., Aramini, J., Swapna, G. V. T.; Montelione, G. T. (2003) Automated protein fold determination using a minimal NMR constraint strategy. Protein Science 2003, 12: 1232-1246.) and certain powerful membrane protein structure analysis methods (Arora, A., Abildgaard, F., Bushweller, J. H., and Tamm, L. K. (2001). Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8, 334-338; Fernandez, C., Hilty, C., Wider, G., Guntert, P., and Wuthrich, K. (2004). NMR structure of the integral membrane protein OmpX. J Mol Biol 336, 1211-1221; Fernandez, C., Hilty, C., Wider, G., and Wuthrich, K. (2002). Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy. Proc Natl Acad Sci USA 99, 13533-13537; Fernandez, C., and Wuthrich, K. (2003). NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Lett 555, 144-150; Sorgen, P. L., Cahill, S. M., Krueger-Koplin, R. D., Krueger-Koplin, S. T., Schenck, C. C., and Girvin, M. E. (2002a). Structure of the Rhodobacter sphaeroides light-harvesting 1 beta subunit in detergent micelles. Biochemistry 41, 31-41; Sorgen, P. L., Hu, Y., Guan, L., Kaback, H. R., and Girvin, M. E. (2002b). An approach to membrane protein structure without crystals. Proc Natl Acad Sci USA 99, 14037-14040; Tamm, L. K., Abildgaard, F., Arora, A., Blad, H., and Bushweller, J. H. (2003). Structure, dynamics and function of the outer membrane protein A (OmpA) and influenza hemagglutinin fusion domain in detergent micelles by solution NMR. FEBS Lett 555, 139-143)).
There are three principle approaches for membrane protein structure analysis by NMR. The first approach is solution-state NMR, which can be used to determine three-dimensional structures of detergent-solubilized membrane proteins using conventional triple-resonance NMR methods with sensitivity-enhanced Transverse Relaxation Optimized Spectroscopy (hereinafter “TROSY”) detection methods (Arora, A., Abildgaard, F.; Bushweller, J. H., and Tamm, L. K. (2001). Structure of outer membrane protein A transmembrane domain by NMR spectroscopy. Nat Struct Biol 8, 334-338; Fernandez, C., Hilty, C., Wider, G., Guntert, P., and Wuthrich, K. (2004). NMR structure of the integral membrane protein OmpX. J Mol Biol 336, 1211-1221; Fernandez, C., Hilty, C., Wider, G., and Wuthrich, K. (2002). Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy. Proc Natl Acad Sci USA 99, 13533-13537; Fernandez, C., and Wuthrich, K. (2003). NMR solution structure determination of membrane proteins reconstituted in detergent micelles. FEBS Left 555, 144-150; Sorgen, P. L., Cahill, S. M., Krueger-Koplin, R. D., Krueger-Koplin, S. T., Schenck, C. C., and Girvin, M. E. (2002a). Structure of the Rhodobacter sphaeroides light-harvesting 1 beta subunit in detergent micelles. Biochemistry 41, 31-41; Sorgen, P. L., Hu, Y., Guan, L., Kaback, H. R., and Girvin, M. E. (2002b). An approach to membrane protein structure without crystals. Proc Natl Acad Sci USA 99, 14037-14040; Tamm, L. K., Abildgaard, F., Arora, A., Blad, H., and Bushweller, J. H. (2003). Structure, dynamics and function of the outer membrane protein A (OmpA) and influenza hemagglutinin fusion domain in detergent micelles by solution NMR. FEBS Lett 555, 139-143). The methods under this approach require the use of 2H, 13C, 15N-enriched membrane protein samples with 13C—1H methyl labels.
The other two approaches are two methods of solid-state NMR, which have been successfully applied to membrane protein structure analysis. One of these approaches, Oriented Solid-State NMR, uses molecular orientation to overcome the line-broadening effects of dipolar coupling and chemical shift that otherwise complicate solid-state NMR spectra of proteins. Pioneered for applications to membrane proteins, samples of lipid bilayers or bicelles are statically-oriented in a special NMR probe, providing a high resolution NMR spectrum that includes information about interatomic bond orientations. This approach, while still under development, has already been used to determine three-dimensional structures of small membrane proteins in lipid bilayers (De Angelis, A. A., Howell, S. C., Nevzorov, A. A., and Opella, S. J. (2006). Structure determination of a membrane protein with two trans-membrane helices in aligned phospholipid bicelles by solid-state NMR spectroscopy. J Am Chem Soc 128, 12256-12267; Kim; S., Quine, J. R., and Cross, T. A. (2001). Complete cross-validation and R-factor calculation of a solid-state NMR derived structure. J Am Chem Soc 123, 7292-7298; Marassi, F. M., and Opella, S. J. (2002). Using pisa pies to resolve ambiguities in angular constraints from PISEMA spectra of aligned proteins. J Biomol NMR 23, 239-242; Marassi, F. M., and Opella, S. J. (2003). Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints. Protein Sci 12, 403-411; Opella, S. J. (2003). Membrane protein NMR studies. Methods Mol Biol 227, 307-320; Park, S. H., Mrse, A. A., Nevzorov, A. A., Mesleh, M. F., Oblatt-Montal, M., Montal, M., and Opella, S. J. (2003). Three-dimensional structure of the channel-forming trans-membrane domain of virus protein “u” (Vpu) from HIV-1. J Mol Biol 333, 409-424; Valentine, K. G., Mesleh, M. F., Opella, S. J., Ikura, M., and Ames, J. B. (2003). Structure, topology, and dynamics of myristoylated recoverin bound to phospholipid bilayers. Biochemistry 42, 6333-6340; Zeri, A. C., Mesleh, M. F., Nevzorov, A. A., and Opella, S. J. (2003). Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy. Proc Natl Acad Sci USA 100, 6458-6463).
The second solid-state NMR approach, Magic Angle Spinning (hereinafter “MAS”) NMR, provides another method for narrowing the broad lines of solid-state NMR samples by minimizing the effects of dipolar coupling by rapidly spinning the solid sample at a special orientation (54.7°) relative to the applied magnetic field. MAS methods have been further developed to the point where it is now possible to obtain complete resonance assignments and three-dimensional structures of small proteins in the solid state, including membrane proteins (Astrof, N. S., Lyon, C. E., and Griffin, R. G. (2001). Triple resonance solid state NMR experiments with reduced dimensionality evolution periods. J Magn Reson 152, 303-307; Castellani, F., van Rossum, B., Diehl, A., Schubert, M., Rehbein, K., and Oschkinat, H. (2002). Structure of a protein determined by solid-state magicangle-spinning NMR spectroscopy. Nature 420, 98-102; Castellani, F., van Rossum, B. J., Diehl, A., Rehbein, K., and Oschkinat, H. (2003). Determination of solid-state NMR structures of proteins by means of three-dimensional 15N-13C-13C dipolar correlation spectroscopy and chemical shift analysis. Biochemistry 42, 11476-11483; Igumenova, T. I., McDermott, A. E. Zilm, K. W., Martin, R. W., Paulson, E. K., and Wand, A. J. (2004a). Assignments of carbon NMR resonances for microcrystalline ubiquitin. J Am Chem Soc 126, 6720-6727; Igumenova, T. I., Wand, A. J., and McDermott, A. E. (2004b). Assignment of the backbone resonances for microcrystalline ubiquitin. J Am Chem Soc 126, 5323-5331; Jaroniec, C. P., MacPhee, C. E., Bajaj, V. S., McMahon, M. T., Dobson, C. M., and Griffin, R. G. (2004). High-resolution molecular structure of a peptide in an amyloid fibril determined by magic angle spinning NMR spectroscopy. Proc Natl Acad Sci USA 101, 711-716; Krabben, L., van Rossum, B. J., Castellani, F., Bocharov, E., Schulga, A. A., Arseniev, A. S., Weise, C., Hucho, F., and Oschkinat, H. (2004). Towards structure determination of neurotoxin II bound to nicotinic acetylcholine receptor: a solid-state NMR approach. FEBS Left 564, 319-324; Petkova, A. T., Baldus, M., Belenky, M., Hong, M., Griffin, R. G., and Herzfeld, J. (2003). Backbone and side chain assignment strategies for multiply labeled membrane peptides and proteins in the solid state. J Magn Reson 160, 1-12; Rienstra, C. M., Tucker-Kellogg, L., Jaroniec, C. P., Hohwy, M., Reif, B., McMahon, M. T., Tidor, B., Lozano-Perez, T., and Griffin, R. G. (2002). De novo determination of peptide structure with solid-state magic-angle spinning NMR spectroscopy. Proc Natl Acad Sci USA 99, 10260-10265).
Solid-state NMR has tremendous potential for providing three-dimensional structures of many membrane proteins that cannot be crystallized. Oriented Solid-State NMR experiments are particularly well-suited for determining structures of helical membrane proteins, and MAS experiments, which can identify dipolar interactions between backbone atoms in adjacent beta strands, are especially well-suited for beta-type membrane structures, though it may also be possible to determine structures of alpha-helical proteins with these new methods.
NMR has special value in structural genomics efforts for rapidly characterizing the “foldedness” of specific protein constructs (Kennedy, M. A., Montelione, G. T, Arrowsmith, C. H., and Markley, J. L. (2002). Role for NMR in structural genomics. J Struct Funct Genomics 2, 155-169; Montelione, G. T (2001). Structural genomics: an approach to the protein folding problem. Proc Natl Acad Sci USA 98, 13488-13489). The dispersion and line shapes of resonances measured in one-dimensional 1H-NMR and two dimensional 15N—1H or 13C—1H correlation spectra provide “foldedness” criteria with which to define constructs and solution conditions that provide folded protein samples (see FIG. 1). The required isotopic enrichment with 15N is relatively inexpensive, and the two-dimensional 15N—1H correlation spectra can be recorded in tens of minutes with conventional NMR systems.
An E. coli Single Protein Production (hereinafter “SPP”) bacterial expression system has been previously described that utilizes a combination of attributes—cold-inducible promoters, low temperature, induction of the mRNA-specific endoribonuclease MazF causing host cell growth arrest, and culture condensation—to facilitate stable, high level protein expression (almost 30% of total cellular protein) without background protein synthesis (Suzuki, M., Roy, R., Zheng, H., Woychik, N., and Inouye, M. (2006). Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem 281, 37559-37565; Suzuki, M., Zhang, J., Liu, M., Woychik, N. A., and Inouye, M. (2005). Single protein production in living cells facilitated by an mRNA interferase. Mol Cell 18, 253-261). This expression system has been shown to provide specific labeling with selenomethionine and fluorophenylalanine (Suzuki, M., Roy, R., Zheng, H., Woychik, N., and Inouye, M. (2006). Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem 281, 37559-37565). Moreover, using an optimized SPP vector, exponentially growing cultures can be condensed 40-fold without significantly affecting protein yields (Suzuki, M., Roy, R., Zheng, H., Woychik, N., and Inouye, M. (2006). Bacterial bioreactors for high yield production of recombinant protein. J Biol Chem 281, 37559-37565). This has the potential to lower sample labeling costs to a small percentage of the cost of traditional isotope-labeling experiments.
The compositions, systems, and methods of the present invention provide effective means to screen conditions for membrane protein purification, membrane protein structure analysis, and to determine three-dimensional protein structures using deuterium-decoupled NMR methods suitable for rapid structure analysis and analysis of large protein structures. The present invention is also advantageous in that it provides means for production of deuterated protein samples at reduced cost.